Nitric oxide (.NO) is an essential bioactive molecule that mediates a variety of actions, such as vasodilation, neurotransmission, and host defense. However, increased .NO production is often associated with the pathogenesis of various disorders, including cancer (Moncada et al, 1991; Kerwin et al, 1995). .NO is produced endogenously by a family of enzymes known as .NO synthases (NOS). Only the inducible isoform (iNOS) produces .NO concentrations in the micromolar range (Nathan, 1997), which is high when compared with the pico- to nanomolar concentrations produced by neuronal (nNOS) and endothelial (eNOS) isoforms (Brovkovych et al, 1999). iNOS is highly active in induced macrophages during chronic and acute inflammation causing excessive nitrosation (covalent attachment of nitroso group to thiols or amines) of proteins, lipids and nucleic acids—a condition known as nitrosative stress.
Recent studies indicate that the increases NOS expression and activity may contribute to tumor development or progression. High levels of .NO synthesis were observed in human gynecological (Thomsen et al, 1994), prostate (Klotz et al, 1998), breast (Thomsen et al, 1995), colon (Ambs et al, 1998), and central nervous system tumors (Cobbs et al, 1995). High levels of .NO are also observed in the most common chronic inflammatory diseases of digestive tract, which predispose individuals to cancer (Wilson et al, 1998; Mannick et al, 1996; Al-Mufti et al, 1998). Furthermore, most infectious bacteria produce a significant amount of NO endogenously and also become a subject to a macrophage-derived NO.
.NO is chemically unreactive toward most bioorganic compounds, but it spontaneously auotoxidizes to yield the highly reactive species, N2O3— the actual nitrosating agent (Williams, 1997; Kharitonov et al, 1995; Grisham et al, 1999) (FIG. 1A). Nitrosation of biological substrates occurs with high efficiency during nitrosative stress in body fluids and tissues (Grisham et al, 1999; Ischiropoulos, 1998). To explain the mechanism of nitrosation in vivo and particularly under noninflammatory conditions when the concentration of free NO is low, the mechanism of micellar catalysis of NO oxidation has been put forward (Liu et al, 1998; Nedosapasov et al, 2000). NO and O2 are both hydrophobic molecules, areas of high hydrophobicity can act as a “sponge” to sequester them from the surrounding aqueous phase. High local concentrations of NO and O2 in a hydrophobic phase, e.g. within lipid membranes, can significantly accelerate NO oxidation and N2O3 formation (Liu et al, 1998; Nedosapasov et al, 2000). Recently, we demonstrated that such micellar catalysis of NO oxidation occurs within the hydrophobic cores of various soluble proteins (Nedosapasov et al, 2000) (FIG. 1B). For example, serum albumin can accelerate the formation of N2O3 more than 15,000 times (Rafikova et al, 2001).
FIG. 1 shows micellar catalysis of .NO oxidation and nitrosation of biological substrates. FIG. 1 shows the third order reaction of .NO with O2 (k=6×106 M−2 sec2) (Wink et al, 1994). FIG. 1 shows hydrophobic compartments (micelles) formed by a protein globule accumulate .NO and O2 from aqueous solution thus accelerating the formation of reactive nitrosating species, N2O3. N2O3 can react with water only at the surface of the protein (via intramolecular NO+ transfer) to form nitrite (NO2−). At the same time, various nucleophiles (e.g. thiols [SH]) can penetrate the protein interior or exist already inside and be accessible for nitrosation (Nedosapasov et al, 2000).